Heat-shock microscope and two-photon fluorescent thermometry

The key optical components required for infrared (IR) laser heat shock:
 

1. An IR laser at the wavelength of around 1342 nm
2. A variable neutral-density filter to adjust IR laser power
3. A pair of lenses (with anti-reflection coatings for IR wavelength) to expand the IR laser beam
4. A pair of scanning galvanometer mirrors to x-y scan the IR laser beam during heat shock
5. An objective with high transmittance for the IR laser (1342 nm)
6. A power meter to measure the IR laser power
7. Other components (e.g. mirrors) to direct the IR laser beam into the microscope system
8. Fluorescent thermometry module (optional): a spectroscope for temperature calibration in the heat-shocked cells/tissues
 

Tips:

1. Expand the IR laser beam to fill the rear aperture of the objective used for heat shock.
2. Comparing to single-spot heat shock (fix the laser on a single spot in cells), the scanning heat shock (scan the IR laser over the targeted cell) can avoid thermal injury to cells.
3. The focal plane of the IR laser should be co-localized with other light beams (e.g. fluorescence excitation lasers) in the microscope.
4. Check the position of the focal point of the IR laser in x, y, and z-axis to make sure the IR laser is precisely targeted on the cells. The methodology for visualizing the IR laser focusing has been illustrated in the Materials and Methods of the paper.
5. Measure the IR laser power on the sample plane using the power meter and calibrate the optical transmittance of the whole system. If heat shock fails due to low transmittance, an IR laser with higher output power is required.
6. For cell heat shock in living organisms, the optimal IR laser power for successful heat shock depends on cell types and locations. The fluorescent thermometry can provide objective criteria to optimize the heat shock conditions (power, time, etc.).

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